Immobilized metal chelate chromatography (IMAC) has been used as a technique for protein purification for several years. The principle behind IMAC lies in the fact that many transition metal ions can form coordination bonds between oxygen and nitrogen atoms of amino acid side chains in general and of histidine, cysteine, and tryptophan, in particular. To utilise this interaction for chromatographic purposes, the metal ion must be immobilised onto an insoluble carrier. This can be done by attaching a chelating ligand to the carrier. Most importantly, to be useful, the metal ion of choice must have a significantly higher affinity for the chelating ligand than for the compounds to be purified. Examples of suitable coordinating metal ions are Cu(II), Zn(II), Ni(II), Ca(II), Co(II), Mg(II), Fe(III), Al(III), Ga(III), Sc(III) etc. Various chelating groups are known for use in IMAC, such as iminodiacetic acid (IDA) (Porath et al. Nature, 258, 598-599, 1975), which is a tridentate chelator, and nitrilotriacetic acid (NTA) (Hochuli et al., J. Chromatography 411, 177-184, 1987), which is a tetradentate chelator.
In the field of IMAC much effort has been placed on providing an adsorbent with a high adsorption capacity for recombinant target proteins, e.g. proteins which contain extra histidine residues, so called histidine-tagged proteins. However, the cells and the fermentation broth wherein the recombinant target protein is produced will also contain other proteins produced by the host cell, generally denoted host cell proteins, some of which will also bind to the adsorbent. Thus, there is a need in this field of an IMAC adsorbent, which adsorbs less host cell proteins and/or which presents an improved selectivity allowing selective binding and/or elution of target proteins.
There are several potential advantages that in theory could be attributed to pentadentate chelating ligands. All protein binding to the metal ion should be weakened compared to tri- and tetra-dentate ligands since the number of coordination sites available for a protein molecule is lower, to the extent that most non-tagged proteins may not bind, leading to higher selectivity for histidine-tagged proteins. This could be of particular importance for low-level target protein expression, where competitive displacement of weak, unwanted binders by the strongest binder, namely the histidine-tagged protein, is difficult to use to an advantage at purification. Furthermore, the stronger binding of metal ions will decrease the loss of the ions during chromatography, decrease the risk for contamination of the purified protein with traces of metal ions, and make the chromatography resin reusable without the need for re-charging of metal ions before the next use. Such aspects are especially important for feeds (samples applied to the chromatographic column) like animal cell culture media and buffers that are “aggressive”, i.e., that tend to remove the immobilized metal ions. Also when substances that disturb the purification by interacting with the metal ions are present in feeds and/or buffers, e.g. some disulfide-reducing agents, it should be an advantage to use IMAC resins that have a pentadentate chelator.
U.S. Pat. No. 6,441,146 (Minh) relates to pentadentate chelator resins, which are metal chelate resins capable of forming octahedral complexes with polyvalent metal ions with five coordination sites occupied by the chelator, leaving one coordination site free for interaction with target proteins. It is suggested to use the disclosed chelator resins as universal supports for immobilizing covalently all proteins, using a soluble carbodiimide. More specifically, the disclosed pentadentate chelator resin is prepared by first reacting lysine with a carrier, such as activated SEPHAROSE™. The resulting immobilized lysine is then carboxylated into a pentadentate ligand by reaction with bromoacetic acid.
McCurley & Seitz (Talanta [1989] 36, 341-346: “On the nature of immobilized tris(carboxymethyl)ethylenediamine”) relates to immobilized pentadentate chelator, namely tris(carboxymethyl)ethylenediamine, also known as TED, used as IMAC stationary phases for protein fractionation. The TED resins were obtained by immobilization of ethylene diamine to a carbohydrate support, and subsequent carboxylation to provide the chelating carboxylic groups. The experimental evidence in the article shows that TED-resins prepared accordingly appear to have a mixture of ligands, with ethylenediamine-N,N′-diacetic acid (EDDA), not TED, predominant. The article also reports a large discrepancy between theoretical metal ion binding capacity determined from the nitrogen content and the experimental capacities, which indicate that a large proportion of the ligands are in a form that does not bind metal ions.
EP 1 244 612 (Akzo Nobel) relates to a process of preparing alkylene diamine triacetic acid and derivatives thereof. More specifically, a process is disclosed, which comprises the conversion of alkylene diamine to a salt of alkylene diamine triacetic acid wherein the reaction is carried out in the presence of a polyvalent metal ion and the entire reaction is carried out under hydrolyzing conditions if any of the reactants contain or form nitrile or amide groups. The suggested use of these compounds is in the field of chelating chemistry, such as metal cleaning.
Haner et al., Analytical Biochemistry 138, 229-234 (1984), describe pentadentate chelator resins produced by linking EDTA covalently to amino-agarose. A cobalt complex of EDTA is used for coupling to the polymer resin via carbodiimide linkage. The described use of the resins was the removal of unwanted Ca2+ ions.